Most genetic material in a bacterium exists as giant DNA molecules which are present as the chromosome of the cell. However, a certain amount of the genetic material may also be present in the form of smaller closed circular molecules of DNA, known as plasmids.
The portion of the DNA molecule related to a specific hereditary trait is called a gene. By techniques referred to as genetic engineering, it is possible to transfer a gene, which codes for the production of a specific protein, from one microorganism to another. Various workers have used these techniques to develop microorganisms which are superior producers of certain proteins such as enzymes.
It has been discovered that plasmids, which contain a series of genes linked together in the form of a circle, can be removed from the cells of one microorganism and inserted into the cells of another microorganism with comparative ease. Plasmids can also be used as vectors to carry new genetic material into a host organism. This is accomplished by first cutting the plasmid with an enzyme, known as a restriction endonuclease, that opens the circle of DNA. A fragment of foreign DNA, containing the desired gene, is inserted into the place where the DNA circle was cut. The circle is reformed by treatment with another enzyme known as a DNA ligase. The recombined plasmid, a new circular DNA molecule, contains the genes of the original plasmid plus the new gene from the piece of DNA which was inserted. This plasmid can be introduced into a host microorganism. The plasmid containing the new gene is then reproduced in the host microorganism and becomes part of its genetic material. The microorganism containing the recombined plasmid then produces the proteins coded for by the genes of this plasmid.
One microorganism that is easily grown on a commercial scale is Bacillus subtilis, hereafter abbreviated B. subtilis. For this reason, researchers have sought to find plasmid vectors which can be inserted into B. subtilis and which will multiply in that host. It is important that the plasmid vector contain some markers which permit easy identification of the presence of that plasmid in the host. Furthermore, it should be possible to insert DNA fragments into the vector without altering its ability to replicate in the B. subtilis.
Several workers have described either naturally-occurring or synthetic plasmids which replicate in B. subtilis. These include Kreft, et al, Molec. Gen. Genet., 162, 59-67 (1978); Keggins, et al, Proc. Natl. Acad. Sci., U.S.A., 75, 1423-1427 (1978); Gryczan, et al, Proc. Natl. Acad. Sci., U.S.A., 75, 1428-1432 (1978); and Gryczan, et al, J. Bacteriology, 141, 246-253 (1980). The plasmid vectors disclosed by these workers contain antibiotic resistance markers to permit their identification in a host.
The present invention is directed to a new synthetic plasmid vector having two antibiotic resistance markers. This vector can be combined with genetic material to form hybrid plasmids that are readily inserted into and maintained in the industrially important microorganisms of the species B. subtilis.